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G Model CBI-6092; No. of Pages 9 ARTICLE IN PRESS Chemico-Biological Interactions xxx (2010) xxxxxx Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint Hprt mutant frequency and p53 gene status in mice chronically exposed by inhalation to benzene Richard J. Albertini a,, Stephen A. Judice a, Leslie Recio b, Vernon E. Walker a a Department of Pathology, University of Vermont, Burlington, VT 05404, USA b Integrated Laboratory Systems Inc., Genetic and Cellular Toxicology Program, Research Triangle Park, NC 27709, USA article info Article history: Available online xxx Keywords: Benzene Hprt Loss of heterozygosity Lymphocytes Gene mutation p53 heterozygous mice abstract Hprt mutant frequency and p53 gene status were assessed in wild-type and p53 heterozygous (p53+/-) mice exposed chronically by inhalation to benzene. Benzene exposures to 100 ppm for 6 h on MondayFriday, 100 ppm for 10 h on MondayWednesdayFriday, or 200 ppm for 5 h on MondayWednesdayFriday yielded the same total exposures (concentration time) of 3000 ppm h/week. Hprt mutations in splenic T-lymphocytes were significantly increased in all benzene groups, ranging from 3.8- to 8.0-fold greater than control values. Wild-type and p53+/- mice were equally susceptible to benzene mutagenesis. Hprt wild-type and mutant isolates from control and exposed animals were examined for TCR gene rearrangements (as markers of in vivo clonality) and for loss of p53 wild-type or mutant alleles. Moderate clonal amplifications were observed among the Hprt mutant but not Hprt wild-type isolates but was not sufficient to account for the increases in Hprt mutant frequencies. Most isolates, whether Hprt wild-type or mutant, retained both p53 alleles in the benzene-exposed p53+/- animals (54% and 63%, respectively, for the Hprt wild-type and mutants). However, 37% of the Hprt wild-type isolates and 46% of the Hprt mutant isolates lost the p53 mutant allele. Only a small percentage of either type of isolate lost the p53 wild-type allele, and this was always in isolates that that previously lost the p53 mutant allele. Loss of the p53 mutant allele was independent of benzene exposure, Hprt status, or 6-thioguanine selection. These findings contrast with the p53 status of thymic lymphomas that had preferentially lost the wild-type p53 allele in some of these same mice. Possible reasons for loss of the mutant p53 allele in the Hprt mutant and wild-type isolates are discussed. 2010 Published by Elsevier Ireland Ltd. 1. Introduction Benzene is a major industrial compound that humans are exposed to as a result of a variety of activities in which it is generated, processed, or used [1,2]. Epidemiological studies have established a relationship between benzene exposure and the development of leukemias, and benzene is classified as a known human carcinogen (Group 1) [1,2]. Benzene also causes various cancers in rodent models. In spite of extensive study, the mechanism(s) underlying benzene-induced neoplasia has yet to be established [3]. In the context of environmental and occupational exposures to chemicals like benzene, biomarkers are non-disease indicators of Abbreviations: CE, cloning efficiency; Hprt, hypoxanthine-guanine phosphoribosyltransferase; MFs, mutant frequencies; M-F, MondayFriday; MWF, MondayWednesdayFriday; PigA, phosphatidylinositol glycan-class A; TCR, T-cell receptor; WT, wild-type. Corresponding author at: 665 Spear St. Burlington, VT 05405, USA. Tel.: +1 802 656 8346; fax: +1 802 899 2583. E-mail address: Ralbert315@aol.com (R.J. Albertini). 0009-2797/$ see front matter 2010 Published by Elsevier Ireland Ltd. doi:10.1016/j.cbi.2009.12.019 exogenous exposures, biological responses, or susceptibility factors of relevance to disease outcomes such as cancer [4,5]. Measures of in vivo mutations at either the gene or chromosome level (aberrations) clearly document non-reversible and heritable alterations in genetic information content in animals or humans, thus, fitting their classification as biomarkers of effect. Current assay techniques allow measures of both gene and chromosome mutations in cancer relevant or "reporter" regions of the genome, the latter serving as surrogates for the former. Among the reporter genes currently used for human biomonitoring are the glycophorin-A gene studied in red blood cells and the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene, the HLA genes and the T-cell receptor (TCR) genes all measured in T-lymphocytes [46]. An emerging assay measures mutations of the phosphatidylinositol glycan-class A (PigA) gene in a variety of cell types of hematological origin [69]. Hprt, TCR and PigA mutations may also be measured in laboratory rodents, monkeys, and humans, allowing for interspecies comparisons [710]. Bacterial transgenes in genetically altered mice provide yet another surrogate marker for cancer gene mutations that might be used to assess effects in various tissues [10]. Please cite this article in press as: R.J. Albertini, et al., Hprt mutant frequency and p53 gene status in mice chronically exposed by inhalation to benzene, Chem. Biol. Interact. (2010), doi:10.1016/j.cbi.2009.12.019 G Model CBI-6092; No. of Pages 9 2 ARTICLE IN PRESS R.J. Albertini et al. / Chemico-Biological Interactions xxx (2010) xxxxxx The potential role of chromosome and gene mutations in the carcinogenic activity of benzene and its metabolites was recently reviewed [3]. Despite the production of many biologically active metabolites of benzene (i.e., hydroquinone, phenol, catechol, benzoquinone, benzenetriol, benzene oxide, muconaldehyde and muconic acid), evidence for specific DNA reactivity has been scant. 32P-postlabeling showed the induction of a N2-(4-hydroxyphenyl)2 -deoxyguansoine-3 -phosphate in HL-60 cells treated in vitro hydroquinone and in bone marrow of mice receiving benzene by intraperitoneal injections [11,12]. However, the in vivo adducts were at low levels and found only following twice-daily high-dose i.p. exposures. Furthermore, in vivo DNA binding studies employing radiolabeled benzene found only low levels of binding without cancer target organ preference [3]. Although in vitro studies of reverse gene mutations in prokaryotic systems have been almost universally negative, mammalian cells have given mixed results for both gene (forward mutations) and chromosomal level events [3]. Tests of metabolites, especially hydroquinone, have provided the most consistently positive results. In vivo, most of the mutagenicity studies in rodents have been positive, with the majority reporting chromosome level mutations [3]. A study in CYP2E1 deficient mice demonstrated the importance of benzene metabolism in the production of micronuclei [13]. Consistent with this report were the findings of positivity for micronucleus induction both in vitro and in vivo by hydroquinone, phenol, catecol and benzenetriol and evidence that benzene metabolites may interact in producing chromosomal damage [3]. In two in vivo studies of gene level mutations in lacI transgenic mice [14,15], benzene-induced alterations in the transgene were almost certainly the result of point-mutations. A single study reported a relationship between subchronic benzene exposure and mutations at the endogenous Hprt gene in splenic lymphocytes of mice, as measured using an autoradiographic assay for Hprt variants [16]. However, because of discordance with cancer bioassay results and lack of a doseresponse effect for Hprt variant frequency [16], this study has been considered to be inconclusive [3]. The purpose of the current study was to measure induction of Hprt mutations in the splenic lymphocytes of mice, using a more definitive T-cell cloning assay, and to assess the status of the p53 tumor suppressor gene in T-cells of wild-type (WT) and p53 heterozygous (p53+/-) mice exposed chronically by inhalation to benzene. For this purpose, spleens were obtained from benzeneexposed animals used in a cancer bioassay of thymic lymphoma induction [17,18], allowing a comparison of surrogate gene mutations and actual tumor production in cells of the same origin in the same group of animals. Furthermore, as p53+/- mice were used in the bioassay, there was opportunity to compare mutagenesis in these animals as compared to WT. The goal was to obtain information on mutation induction by benzene as well as the use of mutations as mechanistic probes. 2. Materials and methods 2.1. Animals, husbandry, and inhalation exposures C57BL/6 WT and C57BL/6 p53+/- mice (the p53+/- construct consists of a partial deletion of intron 4/exon 5 of p53 with an insertion of a neomycin cassette) were obtained from Taconic Farms (Germantown, NY) at 45 weeks of age, and were free of virus titers using standard mouse virus antibody assays. Mice were held for 13 weeks, and acclimated to wire caging within the inhalation chamber for 1 week prior to initiation of benzene exposure. At benzene exposure initiation, mice ages ranged from 6 to 9 weeks. Mice were kept in a reverse 12:12-h light/dark cycle; exposure took place during the light cycle. Each exposure or control group was housed in a separate 8 m3 inhalation chamber, and all mice were individually used in stainless steel wire mesh cages that conformed to federal guidelines. Water (reverse osmosis treated) and commercially available rodent diet were available ad libitum, and feed exposed to benzene was discarded following each exposure period. All procedures involving the use of animals were approved by the Institutional Animal Use and Care Committee. Benzene exposures have been previously described in detail [18]. In brief, WT and p53+/- mice were exposed whole-body for up to 38 weeks in 8 m3 inhalation chambers to target benzene concentrations of 0, 100, or 200 ppm. The benzene-exposed mice consisted of three groups: two at 100 ppm, one at 200 ppm, and one control sham-exposed group at 0 ppm. Start dates for each exposure group were staggered at intervals of 12 weeks apart and no exposures were conducted on the weekend. The control group and one group at 100 ppm benzene were exposed for 6 h/day, 5 days/week on MondayFriday (MF), while the other group at 100 ppm benzene was exposed 10 h/day, 3 days/week on Monday, Wednesday, and Friday (MWF). The group at 200 ppm was exposed 5 h/day, 3 days/week (MWF). These schedules maintained all benzeneexposed groups at an equal exposure level of 3000 ppm h/week. Benzene exposure concentrations were generated by metering known amounts of liquid benzene into a heated j-tube. Benzene vaporized in the j-tube and was carried in a nitrogen stream to the HEPA-filtered chamber air supply where it was diluted to the target concentration. The chamber air supply flowed at -1800 l/min and was conditioned to 72 F and 50% relative humidity. The control chambers (0 ppm) were operated under similar environmental conditions. Benzene exposure concentrations were measured with four calibrated infrared spectrophotometers (MIRAN 1A, the Foxboro Co., Foxboro, MA) with one instrument sampling each chamber. The distribution of benzene was checked at nine locations in each chamber prior to initiation of exposure. The environmental conditions were monitored continuously; 30 min averages were recorded and printed daily. 2.2. Isolation and selection of Hprt mutant T-lymphocytes; diagnosis of thymic lymphomas Due to the staggered state dates for the exposed groups, spleens were collected from benzene-exposed mice for isolation of splenic lymphocytes at 3337 weeks after initiation of the inhalation study (corresponding to 33, 37, and 34 weeks of benzene exposure to 100 ppm MF, 100 ppm MWF, and 200 ppm MWF, respectively) [18]. Reagents for cell culture were the highest grade available from commercial sources and have been listed in detail elsewhere [1921]. At necropsy, spleens were harvested, weighed, and then stored in fresh RPMI 1640 medium on ice for shipment overnight for isolation of splenic lymphocytes. Immediately upon receipt, spleens were `milked' by piercing with a 20-gauge bent needle and then massaging out the contents in RPMI 1640 medium. The general procedures for washing of isolated cells, growth stimulation of mouse T-cells, selection of Hprt mutant T-cell colonies, and calculation of Hprt mutant frequencies (MFs) has previously been described in detail [1921]. Briefly, cells from spleens and thymuses were resuspended in T-flasks with "priming" media (containing rat T-STIM, mouse IL-2, and Con A) overnight at 37 C and 6% CO2. Stimulated lymphocytes were then cultured to generate Hprt mutant T-cell colonies. U-bottom 96-well microtiter plates were seeded with 4 104 cells/well in the presence of 0.5 g 6-thioguanine/ml in 100 l/well of fully supplemented medium. Two 96-well plates were seeded with 2 and 4 cells/well plated in the presence of 3 104 lethally irradiated autologous "feeder" cells/well to determine cloning efficiencies (CEs). Plates were scored for colony Please cite this article in press as: R.J. Albertini, et al., Hprt mutant frequency and p53 gene status in mice chronically exposed by inhalation to benzene, Chem. Biol. Interact. (2010), doi:10.1016/j.cbi.2009.12.019 G Model CBI-6092; No. of Pages 9 ARTICLE IN PRESS R.J. Albertini et al. / Chemico-Biological Interactions xxx (2010) xxxxxx 3 growth with and inverted phase contrast microscope under 20 magnification 810 days later. CEs were calculated based on the Poisson distribution, which allows one to determine the average number of colony-forming units per well ( ), and then Hprt MFs were calculated as the ratio of the mean CE in selective media to that in non-selective media. CEs and MFs for experimental groups were expressed as the mean the standard deviation (SD). Positive colonies were resuspended and placed in 0.5 ml microcentrifuge tubes. Approximately 104 cells were pelleted via centrifugation at 6500 g for 3 min. Growth medium was removed by pipetting and pellets were snap frozen in liquid nitrogen and stored at -70 C until molecular analyses. Thymic lymphomas were diagnosed based on impression smears stained with CMS Protocol HEMA 3 stain set (Biochemical Sciences, Swedesboro, NJ); findings were reviewed by a veterinary pathologist. The tumors consisted predominantly of large round cells with lymphoid appearance and blast T-cell characteristics. 2.3. PCR amplification and DNA sequencing of mouse TCR beta gene mRNA and p53 DNA PCR amplification and DNA sequencing of TCR beta cDNA was carried out directly from pellet lysates. Cell pellets were lysed and reverse transcribed simultaneously using the PerkinElmer reverse transcription kit (#N808-0143). The final reaction component concentrations were as follows: 1 PerkinElmer PCR buffer II, 5 mM MgCl2, 1 mM each dNTP, 2.5 M oligo dT, 10 units RNase inhibitor, 25 units MuLV reverse transcriptase, and 2.5% NP40 final concentrations. Reaction tubes were placed in a Perkin Elmer 9600 and complete one reverse transcription cycle at 25 C for 10 min, 42 C for 15 min, and 99 C for 5 min (to inactivate the MuLV reverse transcriptase enzyme). Following reverse transcription, a single round of PCR was completed using a constant region primer (C beta 5 -GGA TGG TTG CAG ACA GAA CCC CC) at a final concentration of 0.4 mM and a set of 22 different variable TCR beta primers corresponding to the different V beta genes each at a final concentration of 0.2 mM. The reaction was set up as a two-part reaction using PerkinElmer Ampliwax Beads in a 9600 thermocycler. The final reagent concentrations were 0.2 mM dNTP, 1 Boehringer Mannheim PWO buffer (no MgSO4), 2 mM MgSO4, and 0.025 units/l PWO polymerase; 5 l of the reverse transcription reaction was used for the PCR. Samples were run through two initial cycles at 97 C for 1 min, 67 C for 3 min, and 72 C for 1 min; these precycles were followed by 40 cycles at 94 C for 30 s, 66 C for 3 min, and 72 C for 1 min. PCR samples were loaded on a 0.8% agarose gel for electrophoresis, gels were stained with ethidium bromide, and bands were excised from the gel. The products were Gene Cleaned (Bio 101) and sequenced with a C beta primer (5 -GGA GAC CTT GGG TGG AGT CAC). TCR DNA sequences were converted to amino acid sequences for presentation and identification of V beta, CDR3 and J beta gene usage. P53 specific PCR was performed in a Perkin Elmer 9600 Thermocycler. Pellets were lysed in T10E1 with 0.5% tween 20, 0.5% NP40, and 0.1 mg/ml proteinase K for 1 h at 56 C followed by 10 min at 96 C. A hot start PCR was carried out with PerkinElmer PCR gems. The final reaction concentrations were 0.3 mM dNTP, 0.3 mM for primer (5 -CAG CAC TGG GGA GGC CAA AGT GGG), 0.3 mM rev. primer (5 -CTT CCA CCC GGA TAC GAT GCT GGG G), 1 Boehringer Mannheim Expand Long PCR Buffer 2, and 0.5 l Boehringer Mannheim Expand Long PCR enzyme. These primers produced either a single WT p53 band, mutant p53 band, or both. The PCR protocol included 2 cycles of 95 C for 1 min, and 70 C for 3 min, followed by 40 cycles of 94 C for 30 s and 69 C for 3 min. Following PCR, samples were separated on a 0.8% agarose gel, gels were stained with ethidium bromide, and bands were excised from of the gel. Sequencing was performed by direct sequencing of the Gene Cleaned PCR product. A Taq DyeDeoxy Terminator Cycle Sequencing kit (PerkinElmer ABI, Boston, MA) was utilized to amplify the signal for automated sequencing. The new Big Dyes are attached to dideoxy terminators, and mixed with dNTPs, buffer, amplitaq DNA polymerase, 15 l of dsPCR product, and 3.1 pmol/10 l reaction of sequencing primer in accordance with the directions with one deviation. One-quarter volume reactions (1/4) were used to save on materials and sample without loss of signal quality. The reaction mix underwent 1 round of 25 cycles at 96 C for 30 s, 50 C for 15 s, and 60 C for 4 min. The products were purified through packed G50 sephadex columns, dried down, resuspended in a formamideEDTA solution (5:1), vortexed, denatured, and electrophoresed on an ABI automated sequencer Model 373. 2.4. Statistical analyses Statistical significance of the differences in benzene-exposed groups versus sham-exposed control mice, or one exposure group versus another, was first evaluated via one-way ANOVA and the HolmSidak method for multiple comparisons. If the data failed a normality test for homogeneity of variance within each population the one-way ANOVA on Ranks and the Dunn's test were applied. The null hypothesis states that there is no difference between the sham-exposed and individual treatment groups or between differing benzene-exposed groups. Statistical analyses were performed using SigmaStat (SSPSSC, Chicago, Il); p-values < 0.05 were considered significant. 3. Results 3.1. CEs and Hprt MFs in splenic T-cells from control and benzene-exposed mice The actual exposure concentrations for benzene were determined using periodic samplings of the inhalation chamber atmospheres. The average inhalation chamber concentrations of benzene for the nominal 100 ppm MF, 100 ppm MWF, and the 200 ppm MWF exposure groups were 100 0.9 ppm, 100 0.7 ppm, and 200 0.6 ppm, respectively. However, based upon physiologically based pharmacokinetic modeling of benzene metabolism and area under the curve for blood levels of a key metabolite hydroquinone [22], the weekly blood dose of hydroquinone was 160, 150, and 79 M h/L for the respective exposures to 100 ppm MF (30 h/week), 100 ppm MWF (30 h/week), and 200 ppm MWF (15 h/week). Among WT and p53+/- mice, there were no significant differences in the average non-selected CEs in splenic T-cells from groups of control and benzene-exposed mice. The means (and ranges) in CEs for each group are as follows: 16.2 6.7% (7.229%) in WT control mice versus 17.4 5.2% (1123%) in p53+/- control mice, 12.3 5.2% (3.922%) in WT mice versus 14.7 11.3% (6.041%) in p53+/- mice exposed to 100 ppm benzene MF, 10.2 4.5% (4.916%) in WT mice versus 15.8 6.6% (9.324%) in p53+/- exposed to 100 ppm benzene MWF, and 17.1 4.3% in WT mice versus 15.3 6.0% (1021.5%) in p53+/- mice exposed to 200 ppm benzene MWF. Thus, there was no evidence of cytotoxic effects based upon CEs in splenic T-cells from chronic benzene exposures of WT or p53+/- mice. Hprt MFs in control and benzene-exposed mice are shown in Table 1. Background MFs in sham-exposed WT and p53+/- mice were similar (1.2 0.5 10-6 and 1.5 1.1 10-6, respectively) and resembled the spontaneous Hprt MF values previously reported in control mice [2325]. The mutagenic responses among the benzene-exposed groups of mice were elevated in both WT (p = 0.002; one-way ANOVA on ranks and Dunn's test due to an Please cite this article in press as: R.J. Albertini, et al., Hprt mutant frequency and p53 gene status in mice chronically exposed by inhalation to benzene, Chem. Biol. Interact. (2010), doi:10.1016/j.cbi.2009.12.019 G Model CBI-6092; No. of Pages 9 4 ARTICLE IN PRESS R.J. Albertini et al. / Chemico-Biological Interactions xxx (2010) xxxxxx Table 1 Hprt mutant frequency data for individual mice exposed to chamber supply air or benzene. Groups of mice were exposed for up to 38 weeks to chamber air or benzene using three different exposure regimens that resulted in an equal weekly cumulative exposure (3000 ppm h/week). T-cells were isolated from spleen and Hprt MFs measured as described in Section 2. Benzene exposure regimen Hprt MF (10-6)a p53 wild-type (+/+) C57BL/6 mice p53+/- C57BL/6 mice 0 ppm 0.91 0.91 0.90 0.86 0.96 1.5 2.2 0.61 0.68 1.7 3 Mean 1.2 0.5 1.5 1.1 100 ppm 6 h/day, 5 days/week 8.0 7.2 6.7 5.4 4.9 Mean 6.4 1.3 21 15 11 11 7.6 6.8 5.9 5.6 4.2 3.7 9.2 5.4 100 ppm 10 h/day, 3 days/week 19 7.9 7.3 4.0 Mean 9.6 6.5 15 11 8.2 5.6 3.3 8.6 4.6 200 ppm 5 h/day, 3 days/week 6 5.3 3.7 2.8 11 10 8.3 5.4 3.3 3.2 3.3 Mean 4.5 1.5 6.4 3.4 a MF values from four mice in the study were greater than two standard deviations above means for the remaining animals in their respective experimental groups and were considered outliers for calculation of the means and SDs shown above. The outcomes of statistical analyses comparing differences among treatment groups remained the same with or without the inclusion of these four MF values, which included 5.6 10-6 in a p53+/+ control mouse, 7.2 10-6 in a p53+/- control mouse, 48 10-6 in a p53+/+ mouse exposed to 100 ppm benzene MWF, and 88 10-6 in p53+/- mouse exposed to 100 ppm benzene MWF. outlier in mice exposed to 100 ppm benzene MWF) and p53+/- mice (p = 0.04; one-way ANOVA and HolmSidak method), with the increases ranging from 3.8- to 8-fold over background depending upon the exposure group. Pair-wise comparisons showed that the increases in Hprt MFs were significant for each of the three benzene exposure regimens in both WT and p53+/- mice (p-values ranging from 0.006 to 0.02) (Table 1). There were no significant differences in the mutagenic effects observed in WT versus p53+/- mice at 100 ppm MF (p = 0.8), 100 ppm MWF (p = 0.3), or 200 ppm MWF (p = 0.5). The observed MF values suggest that the mutagenic responses were greater in mice exposed to 100 ppm benzene than in those exposed to 200 ppm benzene (Fig. 1). In p53+/- mice exposed to 100 ppm benzene MF and 100 ppm benzene MWF, the mean-induced Hprt MF (i.e., observed MF value - spontaneous control mouse MF value) was 1.6-fold and 1.4-fold higher than the mean-induced mutagenic response in mice exposed to 200 ppm benzene MWF. However, differences in the observed Hprt MFs were Fig. 1. Frequency of Hprt mutations (MF) in splenic T-cells from wild-type (WT) and p53 heterozygous (p53+/-) mice exposed by inhalation to benzene. Mice were exposed for up to 38 weeks to 0 or 100 ppm benzene for 6 h/day on MondayFriday (MF), 100 ppm benzene for 10 h/day on Monday, Wednesday, and Friday (MWF), or 200 ppm benzene for 5 h/day on MWF to achieve an equal exposure level of 3000 ppm h/week. Mutagenic responses in all benzene exposure groups were significantly elevated over spontaneous MF values in control mice, but there were no differences in Hprt MFs observed between differing benzene exposure regimens or between benzene-exposed WT and p53+/- mice. not significantly different among mice in the three benzene exposure regimens, possibly due in part to the limited sample size. 3.2. Relationships between Hprt gene status, TCR gene rearrangement patterns, and/or p53 allele status in T-cell isolates or thymic lymphomas from benzene-exposed p53+/- mice TCR gene rearrangement patterns of T-cell isolates from individual benzene-exposed p53+/- mice can be found in Supplementary data to this report; a total of 147 Hprt mutant and 86 Hprt WT T-cell isolate TCR gene sequences from 16 individual mice are represented. Previous studies have shown that chemical induction of Hprt mutations in T-cells of rodents occurs predominately in preT-cell pools, with mutant cells then trafficking though the thymus and to the peripheral blood and spleen [26,27]. Therefore, most Hprt mutant T-cells in vivo are TCR defined during maturation in the thymus [27]. Post-thymic clonal amplifications of mature Hprt mutant T-cells (recovered from mouse spleens), as recognized by the recovery of two or more Hprt mutant isolates with the identical TCR gene hypervariable (CDR3) sequence, were found only in the Hprt mutant isolates of benzene-exposed mice. Table 2 lists individual benzene-exposed mice that had one or more sets of Hprt mutant T-cell isolates that shared TCR gene identities. Comparison of mouse 642 Hprt mutant isolates M9, M11, M15, M17, and M30, which had the same TCR gene sequence, revealed the occurrence of p53 locus instability as evidenced by progressive loss of alleles in mature T-cells that had undergone post-thymic proliferation. Table 3 lists the TCR gene sequences found in thymic lymphomas of benzene-exposed p53+/- mice as well as spontaneous lymphomas from control mice containing the p53 transgene. Analyses of the spectra of p53 genotypes in lymphomas was found to vary from the spectra of p53 genotypes of peripheral blood T-cell Hprt WT and mutant isolates analyzed in this study. For example, loss of the mutant p53 allele was a relatively frequent event in T-cell isolates, occurring in one or more Hprt WT and mutant isolates from 63% to 69% of 16 benzene-exposed mice, respectively (see Table 2 and Supplementary data). In contrast, among 19 T-cell lymphomas from benzene-exposed mice analyzed in this study, loss of mutant p53 allele occurred in only 11% (2/19) of the lymphomas while both Please cite this article in press as: R.J. Albertini, et al., Hprt mutant frequency and p53 gene status in mice chronically exposed by inhalation to benzene, Chem. Biol. Interact. (2010), doi:10.1016/j.cbi.2009.12.019 G Model CBI-6092; No. of Pages 9 ARTICLE IN PRESS R.J. Albertini et al. / Chemico-Biological Interactions xxx (2010) xxxxxx 5 Table 2 T-cell receptor (TCR) and p53 genotypes in Hprt mutant splenic T-cell isolates from benzene-exposed p53+/- mice. Groups of mice were exposed for up to 38 weeks to benzene using three different exposure regimens that resulted in an equal weekly cumulative exposure (3000 ppm h/week). Column 1 shows the T-cell isolate name; columns 26 give the TCR amino acid sequences; and column 7 shows the p53 allele status of the isolate. Abbreviations: M with a number = mutant isolate number; AA seq = amino acid sequence; WT = p53 wild-type allele; M = p53 mutant allele; LOWT = loss of wild-type p53 allele; LOM = loss of mutant p53 allele; ND = not determined. T-cell isolate # V beta CDR3 J beta p53 genotype Mouse 670 (100 ppm MF) M9 M12 M20 M38 Family 5S2 5S2 8S2 8S2 AA seq CASS CASS CASGD CASGD AA seq RDWGG RDWGG ARTMN ARTMN AA seq DTQYFG DTQYFG TEVFFG TEVFFG Family 2S5 2S5 1S1 1S1 WT/LOM WT/M WT/M WT/M Mouse 642 (100 ppm MF) M9 M11 M15 M17 M30 M28 M36 5S2 5S2 5S2 5S2 5S1 16S1 16S1 CASSL CASSL CASSL CASSL CASSL CASSL CASSL LGV LGV LGV LGV LGV GT GT AETLYFG AETLYFG AETLYFG AETLYFG AETLYFG NSDYTFG NSDYTFG 2S3 LOM/WT 2S3 WT/M 2S3 LOM/WT 2S3 WT/M 2S3 LOM/LOWT 1S2 LOM/WT 1S2 LOM/WT Mouse 455 (100 ppm MWF) M6 M7 6S1 6S1 CASS CASS PGTGGF PGTGGF EQYFG EQYFG 2S7 ND 2S7 ND Mouse 449 (100 ppm MWF) M15 M20 M2B M3 M11B M1B 4S1 4S1 8S3 8S3 15S1 15S1 CASS CASS CAS CAS CGAR CGAR PTTGG PTTGG GTEN GTEN TNFQRKIIF TNFQRKIIF YEQYFG YEQYFG SGNTLYFG SGNTLYFG RS. . . RS. . . 2S7 WT/M 2S7 WT/M 1S3 WT/M 1S3 WT/M 1S4 ND 1S4 ND Mouse 902 (200 ppm MWF) M2 M4 11S1 11S1 CASSL CASSL DWGG DWGG EQYFG EQYFG 2S7 LOM/WT 2S7 LOM/WT WT and mutant p53 alleles were found in 89% (17/19) of the lymphomas (Table 3). However, no loss of the entire WT p53 gene was observed in tumors from these mice (Table 3), as was found in an occasional Hprt WT or mutant T-cell isolate from the same animals (Table 2 and Supplementary data). Data reported here from lymphomas diverge from the overall results of an earlier study [17] where a WT p53 allele loss rate of 89% (24/27) was found with retention of the mutant p53 allele (or transgene) in lymphomas from the same groups of benzene-exposed p53+/- mice. Furthermore, there was a concurrent doubling of the mutant p53 allele in 83% (20/24) of the tumors that had lost the WT p53 allele in the earlier study [17], which was not observed in the current work (Table 3). The disparity between studies is illustrated in Table 3 by the differences in p53 genotypes "determined" (column 7) and "reported" (column 8) in tumor numbers 643, 677, 414, 449, 451, and 455. It should be noted that the TCR gene sequences exhibited by the tumors were not found among the WT or Hprt mutant isolates recovered from the peripheral T-cell population in our study (Tables 2 and 3, and Supplementary data). PCR analysis revealed the pattern of allelic loss as seen in Supplementary data and summarized in Table 4. In benzeneexposed p53+/- mice (all of which developed T-cell lymphomas), 54% of the Hprt mutant isolates retained both p53 WT and p53 mutant alleles, 46% lost the p53 mutant allele, and 6.5% lost both the p53 WT and p53 mutant alleles. This distribution of p53 allele loss can be compared with the distribution observed for the Hprt WT isolates from these same animals, where 63% retained both the p53 WT and p53 mutant alleles, 37% lost the p53 mutant allele, and 9.6% lost both the p53 WT and p53 mutant alleles. Although relatively few T-cell isolates were analyzed in the sham-exposed p53+/- mice, the distribution for retention of both p53 alleles, loss of the p53 mutant allele, and loss of both p53 WT and mutant alleles was 30%, 70%, and 10%, respectively, among the Hprt mutant isolates compared with 60%, 40%, and 20%, respectively, for the Hprt WT isolates. It is noteworthy that the p53 WT allele was never lost alone. Therefore, the percentages of isolates that have lost both p53 alleles are included in the percentages that have lost the p53 mutant allele. In Hprt mutant and WT T-cell isolates recovered from p53 WT mice, a small number (5%) lost both p53 WT alleles in the absence of benzene exposure while the vast majority of isolates retained both p53 alleles. Neither benzene exposure nor 6-thioguanine selection influenced frequencies of p53 allele loss (mutant or WT) in the T-cell isolates. Lineage analyses were possible for in vivo Hprt mutant clonal amplifications found in two different animals (Table 2, mouse 642 T-cell isolates M9, M11, M15, M17, and M30; mouse 670 T-cell isolates M9 and M12). These in vivo clones were established on the basis of their TCR gene rearrangements and characterized by different p53 allele retentions among the different isolates for a given in vivo clone. Among five Hprt mutant isolates with the same TCR sequence in mouse 642, two T-cell isolates retained both the p53 mutant allele and the p53 WT allele, two isolates retained only the p53 WT allele, and one isolate lost both the p53 WT and the p53 mutant allele (Fig. 2A). In two Hprt mutant isolates with the same TCR sequence in mouse 670, one isolate retained both the p53 mutant allele and p53 WT allele while the other isolate had lost the p53 mutant allele (Fig. 2B). Loss of the p53 WT allele was an infrequent event and was the last in the sequence of events when it did occur. Of the groups evaluated in these studies, i.e., control versus benzene-exposed p53+/- mice, Hprt mutant versus Hprt WT T-cell isolates, and 6-thioguanine selected versus non-selected cells, no differences were observed in the rate of p53 mutant allele loss. 4. Discussion The current finding that chronic inhalation of benzene-induced significant increases in Hprt MFs in T-lymphocytes of exposed mice Please cite this article in press as: R.J. Albertini, et al., Hprt mutant frequency and p53 gene status in mice chronically exposed by inhalation to benzene, Chem. Biol. Interact. (2010), doi:10.1016/j.cbi.2009.12.019 G Model CBI-6092; No. of Pages 9 6 ARTICLE IN PRESS R.J. Albertini et al. / Chemico-Biological Interactions xxx (2010) xxxxxx Table 3 T-cell receptor (TCR) and p53 genotypes of thymic lymphomas from control and benzene-exposed p53+/- mice. Groups of mice were exposed for up to 38 weeks to chamber air or benzene using three different exposure regimens that resulted in an equal weekly cumulative exposure (3000 ppm h/week). TCR data for individual tumors (column 2) in differing exposure groups (column 1), displayed as amino acid sequence of the hypervariable CDR3 region (column 5) and adjacent V beta (column 4) and J beta regions (column 6), are listed along with p53 allele status as found in the current study (column 7) and in an earlier study reported by Boley et al. [17]a (column 8). Columns 4 and 6 show the family (in parentheses) and amino acid sequence for the V beta and J beta regions, respectively. Sequence quality is given in degrees sequence heterogeneity in column 3: 1 = a single TCR sequence, 2 = 2 TCR sequences, 3 = mixed TCR sequences but interpretable. TCR sequences that could not be interpreted, possibly because of sample contamination by peripheral blood lymphocytes, are not shown. Abbreviations: WT = p53 wild-type allele; M = p53 mutant allele; MU = mutant (insert) p53 allele LOWT, loss of wild-type p53 allele; LOM, loss of mutant p53 allele; ND, not determined. Exposure regimen Tumor # Sequence quality V beta CDR3 J beta Determined p53 genotype Reported p53 genotypea Control 176 3 227 3 228 3 229 3 230 3 232 3 (1s1) CASSQ (1s1) CASSQ (1s1) CASSQ (1s1) CASSQ (1s1) CASSQ (1s1) CASSQ DGGGV DGGGV DGGGV DGGGV DGGGV DGGGV QDTQYFG (2s5) QDTQYFG (2s5) QDTQYFG (2s5) QDTQYFG (2s5) QDTQYFG (2s5) QDTQYFG (2s5) WT/MU WT/MU WT/MU WT/MU LOWT/LOM WT/MU ND ND ND ND ND ND 100 ppm MF 642 3 643 2 646 1 647 1 660 1 668 1 670 1 677 1 679 1 680 1 (1s1) CASSQ (14s1) CASS (8s2) CAS (4s1) CASSQ (8s2) CASG (1s1) CASSQ (1s1) CASSQ (11s1) CASSL (10s1) CASS (8s2) CASGD DGGGV PGDCE S DRGM QGLA EGTGL AWGDN DGGG HDRGG Y QDTQYFG (2s5) DTQYFG (2s5) QDTQYFG (2s5) DTQYFG (2s5) YAEQFFG (2s1) YEQYFG (2s7) YAEQFFG (2s1) AETLYFG (2s3) AETLYFG (2s3) SYEQYFG (2s7) WT/M WT/LOM LOWT/LOM WT/M WT/M WT/M WT/M WT/M WTb /M WT/M ND Loss WT ND ND ND ND ND Loss WT ND ND 100 ppm MWF 402 414 449 451 455 459 461 2 3 3 1 3 3 1 (10s1) CASS (10s1) CAS (7s1) CASS (8s3) CASSD (?) CAS (1s1) CASSQ (6s1) CAS LEGLGAY RGEGLVRTT YVPNGPGER GWTGGF TATGE DGGGV RPTGED TQYFG (2s5) QYFG (2s5) REQFFG (2s1) EQYFG (2s7) DTQYFG (2s5) QDTQYFG (2s5) TGQLYFG (2s2) WT/M WTb /M WTb /M WT/M WT/M WT/M WT/M ND Loss WT Loss WT Loss WT Loss WT ND ND 200 ppm MWF 902 928 1 1 b Greatly decreased intensity WT band. (6s1) CASS (6s1) CASSI LGQGT SPQGGG YAEQFFG (2s1) TGQLYFG (2s2) WTb /M WT/M ND ND considerably expands earlier limited evidence [1416] that benzene causes gene level mutations in vivo. Surprisingly, however, WT and p53+/- mice were equally sensitive to the mutagenic effects of benzene. Lineage analysis of in vivo Hprt mutant clones revealed the frequent loss of the mutant rather than the WT p53 alleles. This relationship of Hprt mutation induction to p53 status in peripheral blood lymphocytes differs from expectations derived from analyses of the neoplasms induced in the same animals, suggesting a possible difference in the mechanisms underlying induction of surrogate gene mutations in T-cells and induction of T-cell lymphomas. As noted in Section 1, benzene and/or its metabolites are well known inducers of both structural and numerical chromosome aberrations, but evidence for mutation induction at the gene level has been inconsistent [3]. Studies of reverse mutations in prokaryotic systems from benzene exposures have been almost universally negative. By contrast, forward gene mutation assays in mammalian cells have been positive [3], as have studies of lacI mutations in transgenic mice exposed to benzene [14,15]. Results of the present study, using a T-cell cloning assay for measuring MFs, clearly demonstrate that inhalation exposures to leukemogenic levels of benzene induce gene mutations at the endogenous Hprt locus in T-cells of mice. The MF and TCR data combined indicate that it was the number of mutations that were responsible for the increases in Hprt MFs in T-cells of exposed mice Table 4 Patterns in loss of p53 alleles in wild-type (WT) and mutant T-cell isolates from control and benzene-exposed p53+/- mice.a,b. Benzene exposure Hprt status of T-cell isolates Retention of both p53 alleles Loss of p53 mutant allele Loss of both p53 mutant and WT allelesc No 10 WT isolates 6 (60%) No 10 mutant isolates 3 (30%) Yes 73 WT isolates 46 (63%) Yes 124 mutant isolates 67 (54%) 4 (40%) 7 (70%) 27 (37%) 57 (46%) 2 (20%) 1 (10%) 7 (8%) 8 (6.5%) a The mutant allele is present because of the insertional disruption and partial deletion of one copy of p53, to accompany a single functional copy, in creating the p53 haploinsufficient (+/-) mouse. Groups of mice were exposed for up to 38 weeks to chamber air or benzene using three different exposure regimens that resulted in an equal weekly cumulative exposure (3000 ppm h/week) (see Section 2). b Lineage analysis is possible for only two in vivo in Hprt mutant clones. In both, the Hprt mutations arose in cells that had retained both p53 alleles with subsequent loss of the p53 mutant allele (and loss of the p53 wild-type allele at a later stage in one case). Note that loss of p53 wild-type (WT) allele occurred only in clones that first had loss of the p53 mutant allele. c In this context, loss of both p53 mutant and wild-type alleles in column 5 is a subcategory of column 4. Therefore, of the 10 wild-type clones obtained from mice that were not treated with benzene, six (60%) retained both p53 alleles while four (40%) lost the mutant inserted allele. Of the latter four, two (20% of the total of 10 isolates) had also lost the wild-type allele. This is the case for all categories of isolates, i.e., p53 wild-type and mutant isolates from non-treated and non-treated animals. The percentages shown in columns 3 and 4 total to 100% of the isolates studied. Please cite this article in press as: R.J. Albertini, et al., Hprt mutant frequency and p53 gene status in mice chronically exposed by inhalation to benzene, Chem. Biol. Interact. (2010), doi:10.1016/j.cbi.2009.12.019 G Model CBI-6092; No. of Pages 9 ARTICLE IN PRESS R.J. Albertini et al. / Chemico-Biological Interactions xxx (2010) xxxxxx 7 Fig. 2. Progressive loss of p53 alleles in propagated peripheral blood Hprt mutant T-cell isolates. (A) Among five Hprt mutant isolates with the same T-cell receptor (TCR) sequence in mouse 642, two T-cell isolates (M17 and M11) contained both the p53 mutant allele and p53 mutant allele, two isolates (M9 and M15) retained only the wild-type allele, and one isolate (M30) lost both the p53+ WT and mutant alleles. (B) In two Hprt mutant isolates with the same TCR sequence in mouse 670, one isolate (M12) contained both the p53 wild-type allele and the p53 mutant allele while the other isolate (M9) had lost the p53 mutant allele. WT = presence of both p53 wild-type and mutant (transgene) alleles; LOM = loss of mutant allele; LOWT = loss of wild-type allele. and not simply the number of mutant cells resulting from extensive clonal proliferations. The finding that mutagenic responses induced in p53+/- mice exposed for 30 h/week to 100 ppm benzene (MF or MWF) was 1.5-fold greater than in p53+/- mice exposed for 15 h/week to 200 ppm benzene (MWF) is consistent with the roughly 2-fold higher levels of circulating hydroquinone, the 2-fold higher levels of accumulation of micronuclei [18], and the 22.7-fold higher incidence of tumors in p53+/- mice similarly exposed to 100 ppm benzene compared to 200 ppm benzene (J. French et al., manuscript in preparation for this special issue). Additional mutagenicity studies performed by our research group employed shorter durations and lower cumulative doses of benzene (i.e., 1, 5, 50 or 200 ppm benzene for 312 days delivering total exposures of 72, 360 and 3600 ppm/h over 24 days); MF measurements, which used the T-cell cloning assay, did not reveal significant induction of Hprt mutations in T-cells of exposed mice (unpublished data). Thus, it is noteworthy that the benzene concentrations used in current studies were high and the duration of the exposures were long, indicating that large cumulative doses (time concentration) are required for the observed mutagenic effect. In light of recent research showing that cells can resolve the effects of some exogenous exposures using pathways that have a predominately error-free resolution capacity [28,29], it is not possible to determine whether or not the results of an earlier mutagenesis study of benzene, which used an autoradiographic assay for Hprt mutations in T-cells of CD-1 mice exposed to low levels of inhaled benzene (<1.0 ppm) [16], reflects differences in methodology or dose-related expression of error-free resolution pathways. The present work illustrates how studies of Hprt mutations in mouse T-cells designed to reflect the in vivo effects of a potential mutagenic exposure also can reveal fundamental biological and toxicological processes, even when these are contrary to expectations. Our studies initially were designed to determine inhalation exposure to benzene induces gene level mutations in mice and, if so, are p53 WT and p53+/- mice differentially susceptible to benzene-induced mutagenesis. If heightened susceptibility to benzene mutagenesis had been observed in the p53+/- mice, we also hoped to determine if this was due to clonally restricted genomic instability in the T-lymphocyte population resulting from loss of the p53 allele. If affirmed, this would then constitute a situation similar to that postulated for the increased susceptibility of p53+/- mice to tumor induction, which is presumed to result from genomic instability resulting from loss of the p53 WT allele in the tumor progenitors [17,30,31]. Our study design was then based on the concept that mutation induction would be more efficient in the p53+/- mice than in the WT animals and that this would be because the loss of WT p53 allele in in vivo clones of cells rendered them genomically unstable. We found, however, that the p53+/- and homozygous WT mice were equally susceptible to benzene-induced mutations. Lineage analysis of Hprt mutant isolates from p53+/- animals revealed the basis for this finding; there were very few clones that had lost the p53 WT allele and, when WT allele loss was observed, it was always preceded by loss of the p53 mutant allele. Lineage analysis was possible for only two Hprt mutant clones derived from the p53+/- animals. In both cases, the Hprt mutations arose in cells that had retained both p53 alleles. Loss of both p53 WT alleles was observed rarely in Hprt mutant isolates from the p53 homozygous WT animals, which may have been an infrequent event in the cells of all animals or may represent infrequent PCR failures in analysis, representing artifacts. This phenomenon indicates that in at least some clones the Hprt mutations were the first somatic events to have occurred. In any case, whether real or artifactual, loss of the WT p53 allele is too infrequent an event to confer a difference in mutagen susceptibility between the p53+/- and homozygous WT mice. These findings are inconsistent with the notion that p53 haploinsufficiency with only a single functional p53 allele renders cells hypermutable because of a dosage effect [17,30,31]. The frequent and asymmetric loss of the mutant p53 allele in the Hprt mutant clones derived from the p53+/- animals initially was unexpected. However, the suggestion that mammalian cells must possess mechanisms to protect against invasion by foreign DNA was made almost 20 years ago [32,33]. This observation is relevant to the current study because the mutant p53 allele in the p53+/- mice used here consists of a partial deletion of p53 intron 4/exon 5 with an insertion of a neomycin cassette containing bacterial DNA [34]. Sophisticated analyses of transgenic mice have revealed that lacI transgenic inserts containing bacterial DNA are eliminated from somatic cells, reducing the numbers of such inserts from expectations in heterozygous animals [35]. That this is a consequence of the bacterial DNA content of the inserts was demonstrated by `mammalianizing' the insert DNA which totally blocked their elimination. Our analyses of Hprt mutant and WT isolates from the p53+/- mice, with lineage analyses when possible of in vivo Hprt mutant clones, allowed a definitive demonstration of the elimination of bacterial DNA from mammalian genomes. Studies with transgenic animals that involve introduction of foreign DNA must be interpreted with this in mind. The p53+/- animals examined for benzene mutagenesis were also used in cancer bioassays, with the production of lymphomas in >80% of the mice [17]. Nearly 90% of the tumors were reported to have lost the WT p53 allele with retention of the mutant allele. The methods (in this earlier study [17]) used included multiplex PCR of the WT and mutant p53 alleles plus characterization of heterozygosity or its loss (LOH) using arrays of closely linked microsatellite markers of mouse chromosome 11. We had the opportunity to study some of the tumors with the methods employed in our work and found retention of both p53 alleles in the majority of instances. There were, however, occasional reductions of the WT allele band and single instances of loss of the p53 mutant allele or of both p53 alleles. Loss of the mutant p53 allele in the tumors occurred Please cite this article in press as: R.J. Albertini, et al., Hprt mutant frequency and p53 gene status in mice chronically exposed by inhalation to benzene, Chem. Biol. Interact. (2010), doi:10.1016/j.cbi.2009.12.019 G Model CBI-6092; No. of Pages 9 8 ARTICLE IN PRESS R.J. Albertini et al. / Chemico-Biological Interactions xxx (2010) xxxxxx in animals where this also was encountered in the lymphocyte isolates recovered from the mutation studies. Our failure to detect loss of only the p53 WT allele in these thymic lymphomas may have been a technical problem due to infiltration with peripheral blood lymphocytes into the tumor sample. Our study of the tumors also included determinations of TCR gene rearrangements in this material. We did detect predominantly monoclonal patterns in most but, again, could not rule out infiltrating peripheral blood lymphocytes producing occasional mixed patterns. None of the TCR rearrangement patterns detected in the tumors were detected in the analyses of either Hprt WT or mutant isolates recovered from the Hprt mutation assays. It is noteworthy that identical TCR gene rearrangement patterns were sometimes observed for different tumors induced in different animals. This intriguing observation may indicate that there are certain cells that are susceptible to progression to thymic lymphoma or, alternatively, that these patterns represent infiltrating peripheral blood lymphocytes that are drawn to the tumors as an immunological response. As these findings were peripheral to the purposes of the study, they must be followed up before definitive statements can be made. Our studies in the Hprt mutant and WT T-lymphocyte isolates from mutation assays described here indicate that the isolated loss of the p53 WT allele in heterozygous mice is a rare event in mature T-cells. Assuming that the thymic lymphomas in the p53+/- mice are mono- or oligo-clonal, and have truly lost this WT p53 allele, suggests that something different must be occurring in the intrathymic environment, perhaps in immature non-differentiated T-cells. The mechanisms of WT allele loss are unknown, although it has been suggested that this is due to the clastogenicity of benzene in exposed animals. The consequences of loss of p53 function in the tumor progenitor cells are also unknown. Studies reported here failed to find a loss of the WT p53 allele in mature T-cells of the heterozygous mice and, therefore, shed no light on this phenomenon. We have, however, learned something about benzene mutagenicity and the persistence in mammalian cells of p53 mutant alleles containing bacterial DNA. Conflict of interest The authors have no conflict of interest to declare. Acknowledgements We acknowledge Drs. Laura N. Healy and Brian Wong for their work on the benzene inhalation study, and Drs. Janice Nicklas and Dale Walker for helpful comments used in revising this report. The inhalation study in benzene-exposed mice, from which materials for the present report were obtained, was performed at the Chemical Industry Institute of Toxicology, now one of the cornerstone institutes at The Hamner Institutes for Health Sciences. The current work was supported, in part, by grants from the Department of Energy (to RJA) and the Health Effects Institute (to VEW). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cbi.2009.12.019. References [1] International Agency for Research on Cancer (IARC), Benzene Overall Evaluations of Carcinogenicity: An Updating of IARC Monographs, vols. 142, Suppl. 7, IARC, Lyon, France, 1987, pp. 120122. [2] International Agency for Research on Cancer (IARC). Benzene, in: Genetic and Related Effects: An Updating of IARC Monographs, vols. 142, Suppl. 7, 1987, IARC, Lyon, France, pp. 9195. [3] Whysner, M.V. Reddy, P.M. Ross, M. Mohan, E.A. Lax, Genotoxicity of benzene and its metabolites, Mutat. Res. 566 (2004) 99130. [4] R.J. Albertini, J.A. Nicklas, J.P. O'Neill, Future research directions for evaluating human genetic and cancer risk for environmental exposures, Environ. Mol. Mutagen. 104 (Suppl. 3) (1996) 503510. [5] R.J. Albertini, HPRT mutations in humans: biomarkers for mechanistic studies, Mutat. Res. 489 (2001) 116. [6] J.A. Swenberg, E. Fryar-Tita, Y.C. Jeong, G. Boysen, T. Starr, V.E. Walker, R.J. Albertini, Biomarkers in toxicology and risk assessment: informing critical doseresponse relationships, Chem. Res. Toxicol. 21 (2008) 253265. [7] R.J. Albertini, PIG-A as a reporter gene for somatic mutations in humans, Environ. Mol. Mutagen. 49 (2008) 539. [8] D. Miura, V.N. Dobrovolsky, Y. Kasahara, Y. Katsuura, R.H. Heflich, Development of an in vivo gene mutation assay using the endogenous Pig-A gene. I. Flow cytometric detection of CD59-negative peripheral red blood cells and CD48negative spleen T-cells from the rat, Environ. Mol. Mutagen. 49 (2008) 614621. [9] D. Miura, V.N. Dobrovolsky, R.A. Mittelstaedt, Y. Kasahara, Y. Katsuura, R.H. Heflich, Development of an in vivo gene mutation assay using the endogenous Pig-A gene. II. Selection of Pig-A mutant rat spleen T-cells with proaerolysin and sequencing of Pig-A cDNA from the mutants, Environ. Mol. Mutagen. 49 (2008) 622630. [10] V.E. Walker, D.A. Casciano, D.J. Tweats, The Viracept-EMS case: impact and outlook, Toxicol. Lett. 190 (2009) 333339. [11] K. Pongracz, W.J. Bodell, Synthesis of N2-(4-hydroxyphenyl)-2 deoxyguanosine-3 -phosphate: comparison by 32P-postlabeling with the DNA adduct formed in HL-60 cells treated with hydroquinone, Chem. Res. Toxicol. 9 (1996) 593598. [12] W.J. Bodell, D.N. Pathak, G. Lvay, Q. Ye, K. Pongracz, Investigation of the DNA adducts formed in B6C3F1 mice treated with benzene: implications for molecular dosimetry, Environ. Health Perspect. 104 (Suppl. 6) (1996) 11891193. [13] J.L. Valentine, S.S.-T. Lee, M.J. Seaton, B. Asgharian, G. Farris, J.C. Corton, F.J. Gonzalez, M.A. Medinsky, Reduction of benzene metabolism and toxicity in mice that lack CYP2E1 expression, Toxicol. Appl. Pharmacol. 141 (1996) 205213. [14] A.H. Mullin, R. Rando, F. Esmundo, D.A. Mullin, Inhalation of benzene leads to an increase in the mutant frequencies of a lacI transgene in lung and spleen tissues of mice, Mutat. Res. 327 (1995) 121129. [15] G.S. Provost, J.C. Mirsalis, B.J. Rogers, J.M. Short, Mutagenic response to benzene and tris(2,3-dibromopropyl)-phosphate in the lambda lacI transgenic mouse mutation assay: a standardized approach to in vivo mutation analysis, Environ. Mol. Mutagen. 28 (1996) 342347. [16] J.B. Ward Jr., M.M. Ammenheuser, V.M. Sadagopa, Ramanujam, D.L. Morris, E.B. Whorton Jr., M.S. Legator, The mutagenic effects of low level sub-acute inhalation exposure to benzene in CD-1 mice, Mutat. Res. 268 (1992) 4957. [17] S.E. Boley, E.E. Anderson, J.E. French, L.A. Donehower, D.B. Walker, L. Recio, Loss of p53 in benzene-induced thymic lymphomas in p53+/- mice: evidence of chromosomal recombination, Cancer Res. 60 (2000) 28312835. [18] L.N. Healy, L.J. Pluta, R.A. James, D.B. Janszen, D. Torous, J.E. French, L. Recio, Induction and time-dependent accumulation of micronuclei in peripheral blood transgenic p53+/- mice, Tg.AC (v-Ha-ras) and parental wild-type (C57BL/6 and FVB/N) mice exposed to benzene by inhalation, Mutagenesis 16 (2001) 163168. [19] Q. Meng, L. Recio, A.A. Reilly, B. Wong, M. Bauer, V.E. Walker, Comparison of the mutagenic potency of 1,3-butadiene at the hprt locus of T-lymphocytes following inhalation exposure of female B6C3F1 mice and F344 rats, Carcinogenesis 19 (1998) 10191027. [20] Q. Meng, T.R. Skopek, D.M. Walker, S. Hurley-Leslie, T. Chen, D.M. Zimmer, V.E. Walker, Culture and propagation of Hprt mutant T-lymphocytes isolated from mouse spleen, Environ. Mol. Mutagen. 32 (1998) 236243. [21] Q. Meng, R.F. Henderson, A.A. Reilly, M. Bauer, T. Chen, R.H. Heflich, V.E. Walker, Mutagenicity of 1,3-butadiene at the hprt locus of T-cells following inhalation exposures of female mice and rats, Mutat. Res. 429 (1999) 107125. [22] C.E. Cole, H.T. Tran, P.M. Schlosser, Physiologically based pharmacokinetic modeling of benzene metabolism in mice through extrapolation from vitro to the in vivo, J. Toxicol. Environ. Health A 62 (2001) 439465. [23] Q. Meng, R.F. Henderson, L. Long, L. Blair, D.M. Walker, P.B. Upton, J.A. Swenberg, V.E. Walker, Mutagenicity at the Hprt locus in T cells of female mice following inhalation exposures to low levels of 1,3-butadiene, Chem. Biol. Interact. 135136 (2001) 343361. [24] Q. Meng, D.M. Walker, J.D. McDonald, R.J. Henderson, M.M. Carter, D.L. Cook Jr., C.L. McCash, S.M. Torres, M.J. Bauer, S.K. Seilkop, P.B. Upton, N.I. Georgieva, J.A. Swenberg, V.E. Walker, Age-, gender-, and species-dependent mutagenicity in T-cells from mice and rats exposed by inhalation to 1,3-butadiene, Chem. Biol. Interact. 166 (2007) 121131. [25] A.D. Tates, F.J. van Dam, C.M.M. van Teylingen, F.A. de Zwart, A.H. Zwinderman, Comparison of induction of hprt mutations by 1,3-butadiene and/or its metabolites 1,2-epoxybutene and 1,2,3,4-diepoxybutane in lymphocytes from spleen of adult male mice and rats in vivo, Mutat. Res. 397 (1998) 2136. [26] V.E. Walker, I.M. Jones, T.L. Crippen, Q. Meng, D.M. Walker, M.J. Bauer, A.A. Reilly, A.D. Tates, J. Nakamura, P.B. Upton, T.R. Skopek, Relationships between chemical exposure, cell loss and proliferation, and manifestation of Hprt mutant T-cells following treatment of preweanling, weanling, and adult B6C3F1 mice with N-ethyl-N-nitrosourea, Mutat. Res. 431 (1999) 371388. [27] S. Judice, T-cell clonality and trafficking among Hprt mutant T-cells isolated from mice treated with N-ethyl-N-nitrosorea, in: Human and Mouse T-Cell Trafficking, Mutation, and Kinetics, University of Vermont, Burlington, VT, 2001, pp. 171202 (doctoral thesis, Chapter 4). Please cite this article in press as: R.J. Albertini, et al., Hprt mutant frequency and p53 gene status in mice chronically exposed by inhalation to benzene, Chem. Biol. Interact. (2010), doi:10.1016/j.cbi.2009.12.019 G Model CBI-6092; No. of Pages 9 ARTICLE IN PRESS R.J. Albertini et al. / Chemico-Biological Interactions xxx (2010) xxxxxx 9 [28] M.S. Eller, T. Maeda, C. Magnoni, D. Atwal, B.A. Gilchrest, Enhancement of DNA repair in human skin cells by thymidine dinucleotides: evidence for a p53mediated mammalian SOS response, Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 1262712632. [29] M.S. Eller, A. Asarch, B.A. Gilchrest, Photoprotection in human skin--a multifaceted SOS response, Photochem. Photobiol. 84 (2008) 339349. [30] J.E. French, G.D. Lacks, C. Trempus, J.K. Dunnick, J. Foley, J. Mahler, R.R. Tice, R.W. Tennant, Loss of heterozygosity frequency at the Trp53 locus in p53deficient (+/-) mouse tumors is carcinogen- and tissue-specific, Carcinogenesis 21 (2001) 99106. [31] J.E. French, R.D. Storer, L.A. Donehower, The nature of the heterozygous Trp53 knockout model for identification of mutagenic carcinogens, Toxicol. Pathol. 29 (Suppl.) (2001) 2429. [32] T.H. Bestor, DNA methylation: evolution of a bacterial immune function into a regulator of gene expression and genome function in higher eukaryotes, Philos. Trans. R. Soc. Lond. B 326 (1990) 179187. [33] W. Doerfier, Patterns of DNA methylation--evolutionary vestiges of foreign DNA activation as a host defense mechanism, Biol. Chem. Hoppe Seyler 372 (1991) 557564. [34] L.A. Donehoer, M. Harvey, B.L. Slagele, M.J. McAruther, C.A. Montgomery Jr., J.S. Butel, A. Bradley, Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumors, Nature (Lond.) 356 (1992) 215 221. [35] H. Scrable, P.J. Stambrook, A genetic program for deletion of foreign DNA from the mammalian genome, Mutat. Res. 429 (1999) 225237. Please cite this article in press as: R.J. Albertini, et al., Hprt mutant frequency and p53 gene status in mice chronically exposed by inhalation to benzene, Chem. Biol. Interact. (2010), doi:10.1016/j.cbi.2009.12.019